title: A new protein-protein interaction sensor based on tripartite split-GFP association.
abstract: Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.
authors.affiliation: INSERM UMR1037, Cancer Research Center of Toulouse, Université de Toulouse, Institut Claudius Regaud, F-31052 Toulouse, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Cabantous
authors.forename: Hau B
authors.initials: HB
authors.lastname: Nguyen
authors.forename: Jean-Denis
authors.initials: JD
authors.lastname: Pedelacq
authors.forename: Faten
authors.initials: F
authors.lastname: Koraïchi
authors.forename: Anu
authors.initials: A
authors.lastname: Chaudhary
authors.forename: Kumkum
authors.initials: K
authors.lastname: Ganguly
authors.forename: Meghan A
authors.initials: MA
authors.lastname: Lockard
authors.forename: Gilles
authors.initials: G
authors.lastname: Favre
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Terwilliger
authors.forename: Geoffrey S
authors.initials: GS
authors.lastname: Waldo
mesh_terms: D000818:Animals; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D057054:Molecular Imaging; D009154:Mutation; D011485:Protein Binding; D015202:Protein Engineering; D025941:Protein Interaction Mapping / Q000379:methods; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D012995:Solubility; D022021:Tacrolimus Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D022021:Tacrolimus Binding Proteins